Proteins detected in HA-Mmr1 IP fraction; In vitro Mmr1 phosphorylation

A capillary reverse-phase HPLC-MS/MS system was composed of a Dionex U3000 gradient pump equipped with a VICI Cheminert valve and Q Exactive equipped with a nano-electrospray ionization (NSI) source (AMR, Tokyo, Japan). The desalted peptides were loaded into a separation capillary C18 reverse-phase column (NTCC-360/100-3-125, 125×0.1 mm, Nikkyo Technos, Tokyo, Japan). Xcalibur 3.0.63 system (Thermo) was used to record peptide spectra over the mass range of m/z 350–1800. Repeatedly, MS spectra were recorded, followed by ten data-dependent high energy collisional dissociation (HCD) MS/MS spectra generated from the ten highest intensity precursor ions. MS/MS spectra were interpreted, and peak lists were generated using Proteome Discoverer 2.2.0.388 (Thermo). Searches were performed using the SEQUEST (Thermo) program against Saccharomyces cerevisiae (SwissProt TaxID = 559292) peptide sequence.